Compositions and methods for reducing the likelihood of implantation failure or miscarriage in recipients of artificial insemination

ABSTRACT

Methods and kits for preventing or reducing the likelihood of implantation failure or miscarriage in a recipient of artificial insemination are provided. The methods include administering into a recipient of artificial insemination in need of such treatment an effective amount of granulocyte colony stimulating factor (G-CSF).

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/238,977, filed Sep. 26, 2008, which is acontinuation application of Ser. No. 11/411,361, now U.S. Pat. No.7,470,662, filed Apr. 24, 2006 which is a continuation application ofPCT/US04/35468, filed Oct. 25, 2004, which claims priority from U.S.Provisional Application Ser. No. 60/514,472, filed Oct. 24, 2003. Theentirety of all of the aforementioned applications is incorporatedherein by reference.

FIELD

The present invention generally relates to methods of preventingimplantation failure or miscarriage during assisted reproduction and, inparticular, to methods for reducing the likelihood of or preventingimplantation failure or miscarriage in recipients of intravaginalinsemination, intracervical insemination, intratubal insemination, andintrauterine insemination.

BACKGROUND

Accompanying the rising age of hopeful parents is the increasing use ofassisted reproductive techniques such as artificial insemination, invitro fertilization (IVF), gamete intrafallopian tube transfer (GIFT),and the like. Artificial insemination (AI) is the process by which spermis placed into the reproductive tract of a female for the purpose ofimpregnating the female by using means other than sexual intercourse. Inhumans, it is used as assisted reproductive technology, primarily totreat infertility but is also increasingly used to enable women withouta male partner to produce children by using sperm provided by a spermdonor. Specifically, in artificial insemination, freshly ejaculatedsperm, or sperm which has been frozen and thawed, is placed in thecervix (intracervical insemination or ICI), in the female's uterus(intrauterine insemination or IUI), in the vagina (intravaginalinsemination or IVI), or in the fallopian tubes (intratubal inseminationor ITI) by artificial means.

The American Society for Reproductive Medicine estimates that thesuccess rate of artificial insemination may be as high as 15 percenteach cycle. Although success rates are higher for other forms offertility treatments, artificial insemination is often one of the firstmethods used. It is less invasive and less expensive than more complexprocedures, such as in vitro fertilization.

Conception, pregnancy and delivery require an intricate and delicateinterplay of physiology and anatomy. Implantation and placentation arecomplex processes involving hormonal, immune, and anatomical changes inthe mother and migration and cellular division of the embryo.

Although pregnancy rates following six cycles of AI or one cycle of IVFcan be as high as 60%, some recipients fail repeatedly. Various uterinepathologies, such as thin endometrium, altered expression of adhesivemolecules and immunological factors, may be the causes for repeatedfailure.

Spontaneous abortion occurs in 15%-50% of diagnosed pregnancies in womenbetween fifteen and forty-five years of age. The formal definition ofrecurrent spontaneous abortion is three or more spontaneous abortions.However, the American College of Obstetrics and Gynecology recommendsthat in women over the age of 35, a thorough workup should be undertakenafter two spontaneous abortions. Approximately 3-4% of women areestimated to fit the formal definition of recurrent spontaneousabortion. The risk of pregnancy loss increases from 15-20% in the firstpregnancy to 40% after one spontaneous abortion.

Although many pregnancies lost in the first trimester are due to fetalcauses; spontaneous abortion, the loss of the products of conceptionprior to the 20^(th) week of pregnancy, is often a disorder of unknownetiology. It has been theorized that spontaneous abortions are a naturalrejection of a fetus with abnormalities incompatible with life; however,this theory has yet to be substantiated.

Risk factors for miscarriage include age, weight and overall health ofthe woman. The prevalence of spontaneous abortion increases withincreasing maternal age, although not with gravidity. The risk begins toincrease rapidly at age 35 years. The risk of spontaneous abortion atage 40 is approximately twice that at age 20. As families are plannedlater and later in life, the frequency of spontaneous abortion will onlyincrease without effective methods of prevention.

Threatened abortion generally presents as cramping and bleeding forwhich treatment is bed rest. This conservative treatment providespalliative care for the mother but does little to alter the outcome. Theuse of hormones is generally contraindicated due to the risk ofcongenital anomalies, including malformation of the vessels of the heartof the embryo and possible genital abnormalities in female offspring.

Preeclampsia and other hypertensive disorders of pregnancy are a leadingglobal cause of maternal and infant illness and death. Symptoms ofpreeclampsia include hypertension, edema and proteinuria with suddenweight gain, headaches and changes in vision. Preeclampsia can preventthe placenta from getting enough blood which can cause low birth weightand other problems for the baby. Although most women with preeclampsiastill deliver healthy babies, some develop eclampsia, a seriouscondition that threatens the life of the mother and the fetus.

The risk of preeclampsia is higher in women carrying multiple babies, inteenage mothers and in women older than age 40. Typically, preeclampsiaoccurs in the late 2nd or 3rd trimesters (middle to late pregnancy)though occasionally it occurs earlier. Preeclampsia affects about 5% ofall pregnancies.

Mild preeclampsia is conservatively treated with strict bed rest andvigilant monitoring of blood pressure. Progression of the disorder istreated with fluids, antihypertensives and magnesium sulfate butdelivery of the fetus provides the only remedy.

In addition to the physical toll of these disorders, the loss of adesired pregnancy takes a tremendous emotional toll on hopeful andexpectant parents. Loss of a pregnancy can lead to feelings ofinadequacy, hopelessness and guilt which can have a devastating effecton individuals and on a marriage.

New methods and compositions are always needed to reduce risksassociated with pregnancy to the health of the mother and fetus.Effective prevention of implantation failure or spontaneous abortion canallow women, especially women at risk, to have successful pregnancies.In particular, effective prevention of these disorders in women whosuffer from infertility can allow women, especially women who seekmedical care in the form of assisted reproduction like artificialinsemination to have successful pregnancies. Prevention of implantationfailure during assisted reproduction allows successful pregnancies,reduces the risks to women, and saves time and money.

SUMMARY

One objective of the present invention is to provide methods,compositions and kits comprising a granulocyte colony stimulating factor(G-CSF) in an amount effective to prevent miscarriage or implantationfailure in an artificial insemination procedure.

One aspect of the present invention relates to a method for preventingor reducing the likelihood of implantation failure or miscarriage in arecipient of artificial insemination. The method comprises administeringto the recipient of artificial insemination an effective amount of acomposition comprising G-CSF.

In one embodiment, the artificial insemination is intrauterineinsemination, intravaginal insemination, intracervical insemination, orintratubal insemination.

In another embodiment, the composition is administered parenterally,administered enterally, or topically.

In another embodiment, the composition is administered by inhalation.

In another embodiment, the composition is administered prior to theartificial insemination.

In another embodiment, the insemination is preceded by a controlledovarian hyperstimulation procedure, and the composition is administeredbefore, during, or after the time of controlled ovarianhyperstimulation.

In another embodiment, the composition is administered daily for one tothirty-five consecutive days.

In another embodiment, the composition is administered daily until theend of the first trimester.

In another embodiment, the composition is administered daily until therecipient presents a normal Th1 response or a normal Th2 response orboth.

In another embodiment, the G-CSF is administered at a dose of between0.1 mcg/kg/day to 600 mcg/kg/day.

In another embodiment, the G-CSF is administered at a dose of between0.5 mcg/kg/day to 300 mcg/kg/day.

In another embodiment, the G-CSF is administered at a dose of between 1mcg/kg/day to 100 mcg/kg/day.

In another embodiment, the G-CSF is administered at a dose of between 1mcg/kg/day to 50 mcg/kg/day.

In another embodiment, the G-CSF is administered at a dose of between 1mcg/kg/day to 10 mcg/kg/day.

In another embodiment, the G-CSF is administered at a dose of between 1mcg/kg/day to 2 mcg/kg/day.

In another embodiment, the G-CSF is administered in the form of anucleotide sequence encoding G-CSF.

In another embodiment, the composition further comprises an additiveselected from the group consisting of cytokines that suppress Th1 immuneresponse, cytokines that enhance Th2 immune response, cytokines thatsupport successful pregnancy through non-immunologic mechanisms,anti-inflammatory agents, and inhibitors of pro-inflammatory cytokines.

In another embodiment, the additive is selected from the groupconsisting of interferon alpha, interferon beta, macrophage colonystimulating factor (M-CSF), granulocyte macrophage colony stimulatingfactor (GM-CSF), leukemia inhibitory factor (LIF), transforming growthfactor beta (TGF-beta), interleukin-1 (IL-1), IL-3, IL-4, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-13, IL-14, IL-15, IL-19, IL-20, IL-21,IL-22, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32,IL-33, and IL-35.

In another embodiment, the composition further comprises apharmaceutically acceptable carrier.

Another aspect of the present invention relates to a kit for preventingor reducing the likelihood of implantation failure and miscarriage in arecipient of artificial insemination. The kit includes an effectiveamount of G-CSF; and a label with instructions for using the G-CSF toprevent or reduce the likelihood of implantation failure andmiscarriage.

Another aspect of the present invention relates to methods of preventingspontaneous abortion by administering an effective amount of G-CSF tothe subject. The methods can be administered to any female subject atrisk for spontaneous abortion. Subjects at risk can be identifiedaccording to the methods described herein or according to methods knownto practitioners in the art. Typically, the subject is in the first orsecond trimester of pregnancy. In certain embodiments, the subject is inthe first 20 weeks of pregnancy. In certain embodiments, the subject isin the first or second months of pregnancy. In certain embodiments, themethods will be administered before pregnancy is achieved.

Another aspect of the present invention provides methods of treating orpreventing preeclampsia and preterm labor by administering to a subjectin need thereof an effective amount of GCSF or an effective amount ofmobilized peripheral blood stem cells. The methods can be administeredto any female subject at risk for preeclampsia or preterm labor.Subjects at risk can be identified according to the methods describedherein or according to methods known to practitioners in the art.Typically, the subject is in the second or third trimester of pregnancy.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of molecular biology, cell biology,immunology, biochemistry, microbiology, gynecology and obstetrics withinthe skill of the art. Such techniques are explained fully in theliterature. All publications, patents and patent applications citedherein, whether supra or infra, are hereby incorporated by reference intheir entirety.

Definitions

As used herein, the following terms shall have the following meanings:

The terms “treat”, “treating” or “treatment” as used herein, refers to amethod of alleviating or abrogating a disorder and/or its attendantsymptoms. The terms “prevent”, “preventing” or “prevention”, as usedherein, refer to a method of barring a subject from acquiring a disorderand/or its attendant symptoms. In certain embodiments, the terms“prevent”, “preventing” or “prevention” refer to a method of reducingthe risk of acquiring a disorder and/or its attendant symptoms.

The term “artificial insemination” refers to an assisted reproductionprocedure where a donor's sperm is deposited into the vagina(intravaginal insemination), cervical canal (intracervicalinsemination), uterine cavity (intrauterine insemination), or fallopiantubes (intratubal insemination) of the recipient. Intrauterineinsemination (IUI) is a subset of artificial insemination in which spermthat have been washed of seminal fluid are placed directly into theuterus to bypass the vagina and cervix. Artificial insemination can beperformed without any fertility drugs on the day of ovulation or can beperformed using fertility drugs to stimulate superovulation (i.e.,release of more than one oocyte per cycle).

The term “spontaneous abortion” refers to delivery or loss of theproduct of conception before the 20th week of pregnancy. The termspontaneous abortion includes but is not limited to miscarriage,threatened abortion, inevitable spontaneous abortion, incompletespontaneous abortion, habitual or recurrent spontaneous abortion ormissed abortion.

The term “miscarriage” is synonymous with spontaneous abortion.

The term “threatened spontaneous abortion” refers to any bleeding orcramping of the uterus in the first 20 weeks of pregnancy.

The term “inevitable spontaneous abortion” refers to bleeding or ruptureof the membranes accompanied by pain and dilation of the cervix.

The term “incomplete spontaneous abortion” refers to expulsion of partof the products of conception or rupture of the membranes.

The term “habitual spontaneous abortion” or “recurrent spontaneousabortion” refers to three or more consecutive spontaneous abortions.

The term “missed abortion” refers to delay in expulsion of a dead fetus.

The term “assisted reproduction” refers to clinical and laboratorytechniques used to enhance fertility in humans and animals, including,but not limited to, in vitro fertilization, FET, ICSI, GIFT, ZIFT,artificial insemination and the like.

The term “in vitro fertilization” refers to the procedure involvingovarian hyperstimulation (optionally), oocyte retrieval from themother-to-be or a donor, fertilization outside the subject's body,embryo culture and embryo transfer. As used herein, embryo transferrefers to the procedure involving transfer to a subject's uterus, of thedeveloping or cleaving embryos or pre-embryos, also termedpreimplantation embryos.

The term “implantation failure” refers to the failure of an embryoproduced by assisted reproduction or through artificial insemination toimplant or to implant normally in the uterus of a recipient subject.

The term “preeclampsia” refers the development of hypertension withalbuminuria or edema typically between the 20th week of pregnancy andthe end of the first week postpartum. Any pregnant subject who developsa blood pressure of 140/90 mm Hg or higher, edema of the face or hands,albuminuria of ≧1+ or whose blood pressure rises by 30 mm Hg systolic or15 mm Hg diastolic (even if less than 140/90 mm Hg) is consideredpreeclamptic.

The term “colony stimulating factor” or “CSF” relates to a growth factorthat promotes and contributes to the maturity of cells, such as,hematopoietic and blood cells. Examples of CSF molecules include, butare not limited to, erythropoietin, G-CSF, GM-CSF, macrophage CSF,interleukin (IL)-3, IL-6 and stem cell factor.

The term “granulocyte-colony stimulating factor” or “G-CSF” refers tocompounds or factors that stimulate proliferation, differentiation,commitment and end cell functional activation of granulocytes in ananimal, including a human subject. G-CSF includes derivatives, mimetics,variants and chemically modified compounds or hybrids thereof asdescribed in U.S. Pat. Nos. 5,399,345; 5,416,195; 5,981,551; 6,166,183and 6,261,550, the contents of which are incorporated by reference inentireties. G-CSF is commercially available under the names Neupogen®(Amgen), Tevagrastim® (Teva), Biograstim® (CT Arzneimittel),Ratiograstim® (Ratiopharm GmbH)), Zarzio® (Sandoz GmbH), FilgrastimHexal® (Hexal AG), Neulasta® (Amgen), Granocyte® and Neutrogin®(Chugai), and Neu-up® (Kyowa Hakko).

The term “granulocyte” refers to a white blood cell containing granules,especially a leukocyte (white blood cell or corpuscle) containingneutrophil, basophil or eosinophil granules in its cytoplasm.

The term “granulocyte/macrophage colony stimulating factor” or “GM-CSF”refers to compounds or factors that stimulate proliferation,differentiation, commitment and end cell functional activation ofmonocytes and granulocytes in an animal, including a human subject.GM-CSF includes derivatives, mimetics, variants and chemically modifiedcompounds or hybrids thereof as described in, for example, U.S. Pat.Nos. 5,895,646; 5,891,429 and 5,908,763; the contents of which areincorporated by reference in entireties. GM-CSF is commerciallyavailable under the trade names Leukine®, Berlex® and Leucomax® (Wyeth).

The term “macrophage colony stimulating factor” or “M-CSF” (also knownas “CSF-1”) refers to compounds or factors that stimulate proliferationor promote survival of monocytes and macrophages in an animal, includinga human subject. M-CSF includes derivatives, mimetics, variants andchemically modified compounds or hybrids thereof as described in, forexample, U.S. Pat. Nos. 5,837,230 and 5,888,495; the contents of whichare incorporated by reference in entireties. M-CSF is commerciallyavailable under the trade name Leukoprol® (Kyowa).

The term “macrophage” relates to a mononuclear, phagocytic white bloodcell that can exit the circulation and enter tissue spaces.

The term “therapeutically effective amount” refers to that amount of anactive agent being administered sufficient to prevent development of oralleviate to some extent one or more of the symptoms of the condition ordisorder being treated.

The term “preterm labor” also known as premature labor, refers to thebeginning of regular contractions that cause the cervix to begindilation and effacement before the 37th week of pregnancy.

The term “effective amount” refers to that amount of an active agentbeing administered sufficient to prevent the disorder or prevent one ormore symptoms of the disorder being treated. In certain embodiments, theterm “effective amount” refers to that amount of an active agent beingadministered sufficient to reduce the risk of the disorder or one ormore symptoms of the disorder.

The term “subject” refers to animals such as mammals, including, but notlimited to, primates (such as humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice and the like. In preferred embodiments, thesubject is a human female.

The term “label” refers to a display of written, printed or graphicmatter on the immediate container of an article, for example the writtenmaterial displayed on a vial containing a pharmaceutically active agent.

The term “labeling” refers to all labels and other written, printed orgraphic matter on any article or any of its containers or wrappers oraccompanying such article, for example, a package insert orinstructional videotapes or computer data storage devices, such as CDsand DVDs, accompanying or associated with a container of apharmaceutically active agent.

The present invention is directed to methods of preventing spontaneousabortion and implantation failure, and methods of treating or preventingpreeclampsia and preterm labor described in detail below.

Methods For Preventing Or Reducing the Likelihood of ImplantationFailure And Miscarriage In A Recipient of Artificial Insemination ByAdministration of G-CSF

In one aspect, the present invention provides methods of preventing orreducing the likelihood of embryo implantation failure and miscarriagein recipients of artificial insemination and particularly in recipientsof intrauterine insemination during assisted reproduction byadministrating to a subject in need thereof an effective amount ofG-CSF.

Artificial insemination (AI) involves depositing a donor's sperm intothe vagina, cervical canal, uterine cavity, or fallopian tubes of therecipient. Intrauterine insemination (IUI) is a subset of artificialinsemination in which sperm that have been washed of seminal fluid areplaced directly into the uterus to bypass the vagina and cervix. IUI canbe performed without any fertility drugs on the day of ovulation or canbe performed using fertility drugs to stimulate superovulation (i.e.,release of more than one oocyte per cycle).

According to many published studies, insemination combined withcontrolled ovarian stimulation (COH) provides better pregnancy rates.COH is most often achieved using clomiphene citrate and human chorionicgonadotropin (HCG) to trigger superovulation. Insemination is performedwithin 2 days of HCG administration. Alternatively, COH can be achievedusing purified or recombinant follicle stimulation hormone with orwithout recombinant or purified luteinizing hormone. Again, this isfollowed by administration of HCG to trigger superovulation.

In a typical AI procedure, the recipient usually is stimulated withmedication to stimulate multiple egg development and the insemination istimed to coincide with ovulation-release of the eggs from the follicles.

On the day of the procedure, a semen specimen is harvested and “washed”in the laboratory (called sperm processing or sperm washing). By thisprocess, the sperm is separated from the other components of the semenand concentrated in a much smaller volume. Various media and techniquescan be used to perform the washing and separation, depending on thespecifics of the individual case and preferences of the fertility doctorand laboratory. The sperm processing takes about 20-60 minutes,depending on the technique utilized.

A speculum is placed in the vagina and the cervical area is gentlycleaned. Then the separated and washed specimen consisting of a purifiedfraction of highly motile sperm is placed either in the vagina(intravaginal insemination, IVI), cervix (intracervical insemination,ICI), higher in to the uterine cavity (intrauterine insemination, IUI),or fallopian tubes (intrafallopian insemination, IFI) using a sterile,thin and soft catheter. Intrauterine insemination has a better successrate than intracervical insemination. Therefore, it is the preferredmethod at some fertility specialist centers and some general obstetricalpractices.

While not intending to be bound by any particular theory of operation,it is believed that a significant percentage of implantation failure andmiscarriage during artificial insemination is caused by or associatedwith inappropriate immune responses in the recipient of the artificialinsemination. In particular, it is believed that subjects at risk forembryo implantation failure or miscarriage present with anoverproduction of T-helper 1 (Th1) cytokines and underproduction ofT-helper 2 (Th2) cytokines. Positive correlations in human and animalmodels have been demonstrated, (see, Kwak-Kim et al., 2003, HumanReproduction 18:767-73, Krishnan et al., 1996, J. Immunol. 156:653-62)but remain controversial (see, Chaouat et al., 2003, J. ReproductiveImmunol. 59:205-17). The Th1 cytokine associated with overproduction canbe interferon-γ (INF-γ). The Th2 cytokines associated withunderproduction can be interleukins 10 and 4 (IL-10 and IL-4).

To prevent or reduce the likelihood of implantation failure, the G-CSFis typically administered before the insemination procedure. Theadministration is continued until implantation of the embryo to theuterine wall is achieved, until the risk of failed implantation isreduced or eliminated, or according to the judgment of a practitioner ofskill in the art.

In certain embodiments, the administration is continued until pregnancyis confirmed. In certain embodiments, G-CSF is administered daily for1-35 consecutive days. In other embodiments, G-CSF is administered dailyuntil the end of first trimester. In certain embodiments theadministration is started before the time of controlled ovarianhyperstimulation or about the time of controlled ovarianhyperstimulation, and continued daily until about 3 days, about 5 days,about 7 days, about 10 days, about 12 days, about 14 days, about 30days, about 45 days, or about 60 days after insemination into thesubject's vagina, cervix, uterus, or fallopian tubes. In certainembodiments, the administration is started about 14 days, 7 days, 5days, 3 days, and 1 day before insemination into the subject's vagina,cervix, uterus, or fallopian tubes. In another embodiment, theadministration is started about the time of controlled ovarianhyperstimulation and continued daily until about the end of the firsttrimester. In another embodiment, the dose is administered for fiveconsecutive days about the time of insemination and continued dailyuntil about 3 days, about 5 days, about 7 days, about 10 days, about 12days, about 14 days, about 30 days, about 45 days, or about 60 daysafter insemination into the subject's vagina, cervix, uterus, orfallopian tubes. In certain embodiments, the administration is continueddaily until the subject presents a normal Th1 immune response or anormal Th2 immune response or both, according to the judgment of apractitioner of skill in the art.

In certain embodiments, an effective amount of G-CSF is administered toa subject at risk of implantation failure or miscarriage. In certainembodiments, a subject at risk is a subject that has failed one or moreartificial insemination procedures. In further embodiments, a subject atrisk is a subject undergoing her first AI procedure. In furtherembodiments, the subject can also be in any other population at risk forfailed embryo implantation or miscarriage as determined by apractitioner of skill in the art. In another embodiment, the subject hashad one or more previous spontaneous abortions. Other subjects at riskinclude those with unusually high Th1 immune responses or unusually lowTh2 immune responses. In further embodiments, the subject can also be inany other population at risk for failed embryo implantation ormiscarriage as determined by a practitioner of skill in the art.

In certain embodiments, the G-CSF is administered to the subject priorto insemination. For instance, the G-CSF is administered to a subjectthat is planning or attempting to become pregnant via artificialinsemination. Thus, the G-CSF can be administered to the mother-to-beduring the controlled ovarian hyperstimulation procedure or prior to theinsemination if no controlled ovarian hyperstimulation procedure isused. The G-CSF can be administered at any time during the artificialinsemination process.

The G-CSF treatment may also be used to prevent or reduce the likelihoodof implantation failure or miscarriage in recipients of other assistantreproductive procedures, such as in vitro fertilization (IVF), frozenembryo transfer (FET), intracytoplasmic sperm injection (ICSI), zygoteintrafallopian transfer (ZIFT), and gamete intrafallopian transfer(GIFT).

In vitro fertilization is an assisted reproduction procedure to overcomefertility problems caused by, for example, tubal disease, endometriosis,oligospermia, sperm antibodies and unexplained infertility. Theprocedure can include controlled ovarian hyperstimulation with“fertility drugs” such as ovarian stimulants like clomiphene citrate andgonadotropin-releasing hormones. Hyperstimulation of the ovaries caninduce growth of the egg (oocyte) and its encasing cells, collectivelyalso termed the ovarian follicles. After sufficient follicular growth,final follicular maturation is induced and oocytes are retrieved orharvested. The oocytes are fertilized in vitro with sperm and theembryos cultured. A small number of embryos, generally 2-4, are thentransferred to the uterus. Despite the transfer of multiple embryos, theterm pregnancy rate is only about 25%.

Frozen embryo transfer (FET) is a procedure that utilizes cryopreservedembryos from a previous cycle of in vitro fertilization or ICSI. Thecryopreserved embryos are thawed and transferred into the uterine cavitythrough a catheter. FET can be done with no medications or with the useof various medications including estrogen and progesterone.

Intracytoplasmic sperm injection (ICSI) involves placing a sperm insidean egg with a microscopic needle, rather than placing many sperm closeto the outside of the egg, as in IVF, in a dish in a lab. Oncefertilization occurs, the resulting embryo is placed in the recipient'suterus.

Gamete intrafallopian transfer (GIFT) is a procedure that combines eggsand sperm in a dish in a lab. The egg/sperm mixture is then surgicallyinjected into the recipient's fallopian tubes using a laparoscope orfiber-thin tube. Fertilization happens inside the recipient's body, andthe embryo implants naturally. Although this procedure was once commonlypracticed, it's rarely used today because the success with IVF is fargreater on average.

To prevent or reduce the likelihood of implantation failure ormiscarriage, the G-CSF is typically administered before the assistedreproduction procedures. The administration is continued untilimplantation of the embryo to the uterine wall is achieved, until therisk of failed implantation or miscarriage is reduced or eliminated, oraccording to the judgment of a practitioner of skill in the art.

In certain embodiments, the administration is started before eggs,sperm, or embryos are transferred into the recipient. In certainembodiments, the administration is continued until pregnancy isconfirmed. In certain embodiments, the administration is started aboutthe time of controlled ovarian hyperstimulation and continued untilabout 3 days, about 5 days, about 7 days, about 10 days, about 12 days,about 14 days, about 30 days, about 45 days, or about 60 days after thetransfer of embryo to the subject's uterus. In certain embodiments, theadministration is started about the time of controlled ovarianhyperstimulation and continued until about the end of the firsttrimester. In another embodiment, the dose is administered for fiveconsecutive days about the time of embryo/gamete transfer. In certainembodiments, the administration is continued until the subject presentsa normal Th1 immune response for a pregnant subject or a normal Th2immune response for a pregnant subject or both, according to thejudgment of a practitioner of skill in the art.

In certain embodiments, the retrieved oocytes or embryos are maintainedand cultured in medium containing G-CSF prior to their transfer to theuterus of the recipient.

Methods of Preventing Spontaneous Abortion With G-CSF

Another aspect of the present invention provides methods of preventingor reducing the likelihood of spontaneous abortion by administering to asubject in need thereof an effective amount of a G-CSF.

While not intending to be bound by any particular theory of operation,it is believed that many instances of spontaneous abortion and recurrentspontaneous abortion are caused or associated with inappropriate immuneresponses in a pregnant subject. In particular, it is believed thatthese subjects at risk for spontaneous abortion and recurrentspontaneous abortion present inappropriate immune cytokines associatedwith a T-helper 1 (Th1) immune response known to those of skill in theart. (See, Kwak-Kim et at, 2003, Hum. Reprod. 18 (4): 676-773.) Incontrast, subjects that have healthy pregnancies typically presentimmune cytokines associated with a T-helper 2 (Th2) immune response. Itis believed that administration of G-CSF can reduce the inappropriateTh1 response and/or increase a T-helper 2 (Th2) immune response in asubject. This invention is thus based, in part, on the discovery thatadministration of G-CSF can shift a subject's immune response towards ahealthy Th2 response during pregnancy and thereby reduce or eliminatethe risk of spontaneous abortion.

The subject can be any mammalian subject at risk for a spontaneousabortion. In particularly preferred embodiments, the subject is a humanfemale. In certain embodiments, the subject has previously had one ormore spontaneous abortions. In further embodiments, the subject haspreviously had two or more spontaneous abortions. In other embodiments,the subject has had recurrent spontaneous abortions, i.e., three or morespontaneous abortions.

In further embodiments, the subject can be any subject in a populationat risk for spontaneous abortion. For instance, the subject can be ahuman female in an age group at risk for spontaneous abortion. Inparticular embodiments, the subject can be a human female greater than35 years of age, greater than 40 years of age or greater than 45 yearsof age. In other particular embodiments, the subject can be a humanfemale less than 20 years of age or less than 15 years of age. However,essentially a woman of any age that presents with a reproductiveinfirmity, such as spontaneous abortion, preeclampsia and preterm labor,is a candidate for obtaining the materials and methods of the instantinvention.

In further embodiments, the subject can also be in any other populationat risk for spontaneous abortion as determined by a practitioner ofskill in the art. In certain embodiments, the subject is threateningabortion. In other embodiments, the subject is obese, morbidly obese,has overall poor health or comorbid conditions that indicate a risk ofspontaneous abortion to the skilled practitioner. In certainembodiments, these conditions can be incompetent cervix, uterineanomalies, hypothyroidism, diabetes mellitus, chronic nephritis, acuteinfection, use of illicit drugs (such as cocaine or crack), immunologicproblems, severe emotional shock and viral infection (especiallycytomegalovirus, herpes virus and rubella) (see Merck Manual 17thedition, 1999, Merck Research Laboratories, Whitehouse Station, N.J., p.2053). In certain embodiments, the subject has had an implantationfailure during a previous assisted reproduction procedure. Othersubjects at risk include those with unusually high Th1 immune responsesor unusually low Th2 immune responses. In further embodiments, thesubject can also be in any other population at risk for spontaneousabortion as determined by a practitioner of skill in the art.

In certain embodiments, the G-CSF is administered to the subject priorto pregnancy. For instance, the G-CSF is administered to a subject thatis planning or attempting to become pregnant. In other embodiments, theG-CSF is administered to a pregnant subject. The G-CSF can beadministered at any time during the first or second trimester ofpregnancy. In preferred embodiments, the G-CSF is administered duringthe first 20 weeks of pregnancy. In other embodiments, the G-CSF isadministered to a subject who is pregnant or attempting to becomepregnant through artificial insemination or natural conception.

Methods For Treating Or Preventing Preeclampsia Or Preterm Labor WithGCSF

In a further aspect, the present invention provides methods of treatingor preventing preeclampsia or preterm labor by administering to asubject in need thereof an effective amount of granulocyte colonystimulating factor.

While not intending to be bound by any particular theory of operation,it is believed that preeclampsia and preterm labor is caused orassociated with inappropriate immune responses in a pregnant subject. Inparticular, it is believed that subjects at risk for preeclampsia orpreterm labor present inappropriate immune cytokines associated with aT-helper 1 (Th1) immune response known to those of skill in the art. Incontrast, subjects that have healthy pregnancies typically presentimmune cytokines associated with a T-helper 2 immune response. It isbelieved that administration of GCSF can reduce the inappropriate Th1response and/or increase a Th2 immune response in a subject. Thisinvention is thus based, in part, on the discovery that administrationof GCSF can shift a subject's immune response towards a healthy Th2response during pregnancy and thereby treat or prevent preeclampsia orpreterm labor.

In the methods of treatment, GCSF is administered to a subjectpresenting one or more signs or symptoms of preeclampsia or pretermlabor. The subject can be any subject that presents any of the signs orsymptoms of preeclampsia during pregnancy such as hypertension, swellingor edema and excessive protein in the urine. For example, the subjectcan be any subject that develops hypertension with albuminuria or edemabetween the 20th week of pregnancy and the end of the 1 st weekpostpartum. Particular subjects include pregnant females who develop ablood pressure of 140/90 mm Hg, edema of the face or hands oralbuminuria of ≧1+ or whose blood pressure rises by 30 mm Hg systolic or15 mm Hg diastolic (even if less than 140/190 mm Hg) between the 20thweek of pregnancy and the end of the 1 st week postpartum. Particularlypreferred subjects are human females.

In the methods of treatment, the GCSF is typically administered untilthe signs or symptoms of preeclampsia or preterm labor are alleviated orreduced as long as the therapeutic benefit outweighs the risk of adverseevents according to the judgment of a practitioner of skill in the art.The dosing can continue as long as the subject displays no toxic effectsof the administration according to the judgment of a practitioner of inthe art. In certain embodiments, the treatment is continued until thesubject presents a normal Th1 immune response for a pregnant subject ora normal Th2 response for a pregnant subject, or both, according to thejudgment of a practitioner of skill in the art.

In the methods of prevention, GCSF is administered to a subject at riskfor developing preeclampsia or preterm labor. The subject can be anymammalian subject at risk for preeclampsia or preterm labor. Subjects atrisk include subjects carrying multiple babies, subjects younger thanage 20 and subjects older than age 40. Further subjects include thosepregnant for the first time (primigravida), subjects with preexistinghypertension and subjects with preexisting vascular disease. Othersubjects at risk include those with unusually high Th1 immune responsesor unusually low Th2 immune responses. In particularly preferredembodiments, the subject is a human female.

In the methods of prevention, GCSF is administered as long as thesubject is at risk for preeclampsia and as long as the therapeuticbenefit outweighs the risk of adverse events and also, so long as notoxicity is observed according to the judgment of a practitioner ofskill in the art. In certain embodiments, GCSF is administered for theduration of the pregnancy. In particular embodiments, administration isprovided in the 2nd and 3rd trimester of pregnancy. In furtherembodiments, administration is continued after delivery for about one,about two, about three, about four, about five, about six, about sevenor about eight weeks post partum. In certain embodiments, the treatmentis continued until the subject presents a normal Th1 immune response fora pregnant subject or a normal Th2 immune response for a pregnantsubject, or both, according to the judgment of a practitioner of skillin the art.

The GCSF can be administered according to any method of administrationknown to those of skill in the art. Preferred methods of administrationinclude subcutaneous administration. Other effective modes ofadministration are described in detail in the sections below.

G-CSF And Formulation

As described in detail above, the present invention provides methods ofadministering an effective amount of granulocyte colony stimulatingfactor (G-CSF) to prevent or reduce the likelihood of spontaneousabortion, implantation failure and miscarriage during and followingartificial insemination.

The G-CSF administered in the methods of the invention can be any G-CSFknown to one of skill in the art without limitation. Thus, a range ofmodifications can be made to the wild-type G-CSF molecules so long asthe known immune system modulating activity of the G-CSF is maintained.There are a number of assays that can be used to ensure that any onemodified G-CSF retains the desired immune system modulating activity.Plural types of G-CSF molecules can be administered in the practice ofthe instant invention. The plural G-CSF molecules can be administeredconcurrently, consecutively, or sequentially. In certain embodiments,the G-CSF can be any G-CSF or any derivative, variant, mimetic,chemically modified version or hybrid thereof, as described in U.S. Pat.Nos. 5,399,345; 5,416,195; 5,981,551; 6,166,183 and 6,261,550, thecontents of which are hereby incorporated by reference in theirentireties. In further embodiments, the G-CSF can be administered in theform of a nucleotide sequence encoding G-CSF or expression vectorsencoding G-CSF described in U.S. Pat. No. 5,422,248, the content ofwhich is hereby incorporated by reference in its entirety. The G-CSF canbe formulated according to any formulation for administration known tothose of skill in the art.

In certain embodiments, the G-CSF is a commercially available G-CSFavailable as a pharmaceutical composition, suitable for administrationto an animal, including a human. Such commercially availablepharmaceutical compositions can be, for example, filgrastim (Neupogen®(Amgen), Tevagrastim® (Teva), Biograstim® (CT Arzneimittel),Ratiograstim® (Ratiopharm GmbH), Zarzio® (Sandoz GmbH), FilgrastimHexal® (Hexal AG), pegfilgrastim (Neulasta®, Amgen), nartograstim(Neu-Up®, Kyowwa) or lenograstim (Neutrogin®, Granocyte®, Chugai).

Filgrastim, nartograstim, and lenograstim are useful for promotingneutrophil proliferation and are generally administered to individualsin need to increased neutrophils, for example, patients undergoingchemotherapy. Filgrastim, nartograstim, and lenograstim are indicatedfor myelosuppressive chemotherapy, bone marrow transplant, peripheralblood progenitor cell collection and severe chronic neutropenia. Offlabel uses include treatment of neutropenia in AIDS patients, aplasticanemia, hairy cell leukemia, myelodysplasia, drug-induced and congenitalagranulocytosis and alloimmune neonatalneutropenia.

The usual treatment of neutropenia associated with myelosuppression is 5mcg/kg/day, once daily either by bolus subcutaneously or short (15-30minute) intravenous infusion or by continuous subcutaneous orintravenous infusion. Administration is once daily starting no earlierthan 24 hours after chemotherapy and continues for 14 days or until theindividual's absolute neutrophil count is 10,000/mm3. For patientsundergoing bone marrow transplant, the usual dose is 10 mcg/kg/dayadministered as an intravenous infusion over 4-24 hours or as acontinuous 24 hour subcutaneous infusion. The first dose is generallyadministered at least 24 hours after chemotherapy and at least 24 hoursafter bone marrow infusion. During recovery, the dose is adjustedaccording to the patient's absolute neutrophil count. Filgrastim dosingfor peripheral blood progenitor cells generally begins at 10 μg/kg/daysubcutaneously either as a bolus or continuous infusion. It isrecommended that filgrastim be given for at least four days beforeleukapheresis and continued until the last leukapheresis procedure.Doses of filgrastim for congenital neutropenia are 5 mcg/kgsubcutaneously twice daily while idiopathic or cyclic neutropenia isgenerally treated with a dose of 5 mcg/kg subcutaneously once daily.

Pegfilgrastim is a monomethoxypolyethylene glycol conjugate offilgrastim. The pharmaceutical composition is commercially available aspreservative free solutions of 10 mg/ml pegfilgratim in prefilledsingle-dose syringes. Pegfilgrastim is indicated to decrease infectionsin patients with febrile neutropenia undergoing myelosuppressivechemotherapy. Recommended dosing is a single 6 mg subcutaneous injectionadministered once per chemotherapy cycle.

In the above-described methods, G-CSF is administered in an effectiveamount, i.e., an amount effective to reduce or eliminate the risk ofimplantation failure or spontaneous abortion. The amount can bedetermined by the skilled practitioner guided by the description hereinand the knowledge in the art. In preferred embodiments, the amount canbe any amount of G-CSF that reduces the Th1 response of the subject. Infurther embodiments, the amount can be any amount sufficient to increaseor initiate a Th2 response in the subject. Assays to determine Th1 andTh2 responses in the subject are well known to those of skill in the art(See e.g., Schust and Hill, 1996, J. Soc. Gynecol Investig. 3:259-61,Xing et al., 2001, Chin. Med. J. 114:921-4, Raghupathy et al., 1999,Cell Immunol. 196:122-30, Mauri et al., 1996, J. Immunol. 26:1511-8,Doncorli et al., 1997, Eur. J. Imm. 27:1451-8, Raziuddin, 1998, J.Rheumatol 25:329-33, Moverare et al., 2000, Allergy 55:171-5). Inparticular embodiments, G-CSF is administered at doses of about 0.1mcg/kg/day to 600 mcg/kg/day, 0.5 mcg/kg/day to 300 mcg/kg/day, 1mcg/kg/day to 100 mcg/kg/day, 1 mcg/kg/day to 50 mcg/kg/day, 1mcg/kg/day to 20 mcg/kg/day, 1 mcg/kg/day to 10 mcg/kg/day, 1 mcg/kg/dayto 2 mcg/kg/day; and about 1.67 mcg/kg/day. In another embodiment, atleast 0.01 mg, at least 0.02 mg, at least 0.05 mg at least 0.1 mg, atleast 0.2 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, at least 5mg, at least 10 mg, at least 25 mg, at least 50 mg, at least 75 mg, atleast 100 mg, at least 125 mg, at least 150 mg, at least 175 mg, atleast 200 mg, at least 300 mg, at least 600 mg or more is administereddaily.

In certain embodiments described above, the present invention providesmethods of administering to a subject in need thereof an effectiveamount of G-CSF as monotherapy. In other embodiments, the presentinvention provides methods of administering to a subject an effectiveamount of G-CSF in combination with at least one additive. Additivesinclude cytokines that suppress Th1 immune response, cytokines thatenhance Th2 immune response, and non-myeloablative immunosuppressiveagents. Additives may also include cytokines that support successfulpregnancy outcome through non-immunologic mechanisms such as stimulationof trophoblast cell proliferation, inhibition of trophoblast cellapoptosis, stimulation of trophoblasat invasion, or stimulation ofangiogenesis. Additives may also include anti-inflammatory agents, andinhibitors of pro-inflammatory cytokines.

Examples of additives include, but are not limited to interferon alpha,interferon beta, macrophage colony stimulating factor (M-CSF),granulocyte macrophage colony stimulating factor (GM-CSF), leukemiainhibitory factor (LIF), transforming growth factor beta (TGF-beta),interleukin-1 (IL-1), IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-13, IL-14, IL-15, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26,IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, and IL-35.

In certain embodiments, additives may include inhibitors ofpro-inflammatory cytokines such as, but not limited to, anti-TNF-alpha,pentoxifyllin, anti-VEGF, anti-CD28, anti-CD80, anti-CD86, anti-CD40L,and the like.

Other additives that may be used with a G-CSF include anti-inflammatoryagents. The anti-inflammatory agent can be one that reduces leukocytepopulations or inhibits leukocyte function. Other anti-inflammatoryagents can be used as well. For example, vitamin D3(1,25-dihydroxycholecalciferol) and analogs thereof can be used. Inanother example, corticosteroids such as prednisone ormethylprednisolone can be used.

Other additives that may be used with a G-CSF are those currently usedto treat recurrent spontaneous abortion, miscarriage, or implantationfailure, such as intravenous Ig and heparin.

The additive can be another CSF, erythropoietin or stem cell factor. TheCSF can be G-CSF, GM-CSF or macrophage CSF.

The G-CSF can be administered according to any method of administrationknown to those of skill in the art. Preferred methods of administrationinclude subcutaneous administration, parenteral administration, enteraladministration, topical administration. G-CSF may also be administeredby inhalation. In the compositions administered, the G-CSF can beformulated in any manner known to those of skill in the art forformulating and administering effective amounts of G-CSF.

Filgrastim or non-glycosylated G-CSF is available as a preservative-freepharmaceutical composition comprising 300 mcg/ml and 480 mcg/ml vials or300 mcg/0.5 ml and 480 mcg/0.5 ml self-injectors. The composition can beadministered subcutaneously without further admixture. Intravenouspreparations require dilution with proper diluent, such as 5% dextrose,diluted to a final concentration of filgrastim of 5 to 15 mcg/ml. Salineis not recommended as a diluent due to product precipitation. Mixturewith albumin is recommended to prevent adsorption to plastic or glassmaterials during preparation and infusion. The final concentration ofhuman albumin should be 2 mg/ml. It is highly recommended thatfilgrastim be refrigerated at 2° to 8° C.

The presently available pharmaceutical composition contains a smallamount of acetate, Tween 80, sodium, and sorbitol. These excipients areused to achieve and maintain characteristics that are physiologicallyacceptable to the body and pharmaceutically practical. Suchcharacteristics include tonicity, osmoticity, osmolality, osmolarity,viscosity and shelf life. Aqueous pharmaceutical compositions of G-CSFwith increased serum half life have been described, for example, in U.S.Pat. No. 5,919,757, incorporated herein by reference in its entirety.

The pharmaceutical compositions can comprise the G-CSF in a salt form.For example, because proteins can comprise acidic and/or basic terminiside chains, the proteins can be included in the pharmaceuticalcompositions in either the form of free acids or bases, or in the formof pharmaceutically acceptable salts. Pharmaceutically acceptable saltscan include, suitable acids which are capable of forming salts with theproteins of the present invention including, for example, inorganicacids such as hydrochloric acid, hydrobromic acid, perchloric acid,nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and thelike; and organic acids such as formic acid, acetic acid, propionicacid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonicacid, succinic acid, maleic acid, fumaric acid, cinnamic acid,anthranilic acid, citric acid, naphthalene sulfonic acid, sulfanilicacid and the like. Suitable bases capable of forming salts with thesubject proteins can include, for example, inorganic bases such assodium hydroxide, ammonium hydroxide, potassium hydroxide and the like;and organic bases such as mono-, di-and tri-alkyl amines (for example,triethyl amine, diisopropyl amine, methyl amine, dimethyl amine and thelike) and optionally substituted ethanolamines (for example,ethanolamine, diethanolamine and the like).

Although commercially available G-CSF is currently administeredsubcutaneously or intravenously, any method of administration thatprovides a therapeutically effective amount of G-CSF can be used in themethods of the invention. In one aspect, G-CSF can be in a variety offorms suitable for any route of administration, including, but notlimited to, parenteral, enteral, topical or inhalation.

Parenteral administration refers to any route of administration that isnot through the alimentary canal, including, but not limited to,injectable administration, i.e., intravenous, intramuscular and the likeas described below. Enteral administration refers to any route ofadministration which is oral, including, but not limited to, tablets,capsules, oral solutions, suspensions, sprays and the like, as describedbelow. For purposes of this invention, enteral administration alsorefers to rectal and vaginal routes of administration. Topicaladministration refers to any route of administration through the skin,including, but not limited to, creams, ointments, gels and transdermalpatches, as described below (see also, Pharmaceutical Sciences, 18thEdition (Gennaro et al., eds., Mack Printing Company, Easton, Pa.,1990).

Parenteral pharmaceutical compositions of the present invention can beadministered by injection, for example, into a vein (intravenously), anartery (intraarterially), a muscle (intramuscularly) or under the skin(intradermally or subcutaneously) or in a depot composition.

Injectable pharmaceutical compositions can be sterile suspensions,solutions or emulsions of the G-CSF in aqueous or oily vehicles. Thecompositions can also comprise formulating agents or excipients, such assuspending, stabilizing and/or dispersing agents. The formulations forinjection can be presented in unit dosage form, in ampules or inmultidose containers, and can comprise added preservatives. In certainembodiments, the pharmaceutical compositions contain buffers such ascitrate, acetate, phosphate, tris (hydroxymethyl) amino methane or THAM(tromethamine).

Depot or sustained release pharmaceutical compositions can be used inthe methods of the invention. For example, continuous release of G-CSFcan be achieved by the conjugation of the G-CSF with a water solublepolymer as described in U.S. Pat. No. 5,320,840. G-CSF may be containedin an inert matrix or device for slow release after implantation of thematrix or device.

Injectable compositions can be pharmaceutically appropriate compositionsfor any route of injectable administration, including, but not limitedto, intravenous, intrarterial, intracoronary, pericardial, perivascular,intramuscular, subdermal, subcutaneous and intraarticular.

Alternatively, the injectable pharmaceutical composition can be providedin powder form for reconstitution with a suitable vehicle, including butnot limited to sterile pyrogen free water, buffer, dextrose solution,etc., before use. To this end, the G-CSF can be lyophilized asappropriate. The pharmaceutical compositions can be supplied in unitdosage forms and reconstituted prior to use in vivo.

For prolonged delivery, the pharmaceutical composition can be providedas a depot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the pharmaceutical composition can be formulated with suitablepolymeric or hydrophobic materials as an emulsion in an acceptable oilor ion exchange resins, or as sparingly soluble derivatives; as asparingly soluble salt form of the G-CSF, or derivative, mimetic orvariant thereof. The G-CSF can be present in an inert matrix or devicefor implantation to achieve prolonged release.

Alternatively, transdermal delivery systems manufactured as an adhesivedisc or patch that slowly releases the active ingredient forpercutaneous absorption can be used. To this end, permeation enhancerscan be used to facilitate penetration of the G-CSF. A particular benefitmay be achieved by incorporating the G-CSF into a transdermal patch.

For oral administration, the pharmaceutical formulations can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., piegelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art (see, Remington'sPharmaceutical Sciences, 18th edition (Gennaro et al., eds.) MackPrinting Company, Pa., 1990).

Liquid pharmaceutical compositions for oral administration can take theform of, for example, solutions, syrups or suspensions, or they can be adry product for constitution with water or other suitable vehicle beforeuse. Such liquid pharmaceutical compositions can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid).

The pharmaceutical compositions can also comprise buffer salts,flavoring, coloring and sweetening agents as appropriate. Pharmaceuticalcompositions for oral administration can be suitably prepared to providecontrolled release of the G-CSF.

Enteral pharmaceutical compositions can be suitable for buccaladministration, for example, in the form of tablets, troches orlozenges. For rectal and vaginal routes of administration, the G-CSF canbe prepared as solutions (e.g. for retention enemas), suppositories orointments. Enteral pharmaceutical compositions can be suitable foradmixture in feeding mixtures, such as, for mixture with totalparenteral nutrition (TPN) mixtures or for delivery by a feeding tube(see, Dudrick et al., 1998, Surg. Technol. Int. VII:174-184; Mohandas etal., 2003, Natl. Med. J. India 16 (1):29-33; Bueno et al., 2003,Gastrointest. Endosc. 57 (4):536-40; Shike et al., 1996, Gastrointest.Endosc. 44 (5):536-40).

For administration by inhalation, the G-CSF can be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator can be formulatedcomprising a powder mix of the compound and a suitable powder base suchas lactose or starch. Inhaled pharmaceutical compositions can be those,for example, described in U.S. Pat. Nos. 5,284,656 and 6,565,841,incorporated herein by reference in their entirety.

The compositions can, if desired, be presented in a pack or dispenserdevice that can comprise one or more unit dosage forms comprising theG-CSF. The pack can, for example, comprise metal or plastic foil, suchas a blister pack. The pack or dispenser device can be accompanied byinstructions for administration.

The pharmaceutical compositions can be for a single, one time use or cancontain antimicrobial excipients, rendering the composition suitable formultiple, extended use with greater shelf stability; for example, amulti-use bottle. In another embodiment, the pharmaceutical compositionof interest can be in unit dose or unit-of-use packages. As known in theart, a unit dose is targeted for a single use. The unit dose form can bein a vial, which can contain a solution or a desiccated form forreconstitution, a pre-filled syringe, a transdermal patch and the like.

As is known to those of skill in the art, a unit-of-use package is aconvenient prescription size, patient ready unit labeled fordistribution by health care providers. The package contains as muchactive ingredient as necessary for a typical treatment regimen.

The pharmaceutical composition can be labeled and have accompanyinglabeling to identify the composition contained therein and otherinformation useful to health care providers and end users. Theinformation can include instructions for use, dose, dosing interval,duration, indication, side effects and other contraindications,warnings, precautions, storage recommendations and the like.

Various embodiments of the pharmaceutical compositions have beendescribed. The descriptions and examples are intended to be illustrativeof the invention and not limiting. Indeed, it will be apparent to thoseof skill in the art that modifications to the pharmaceuticalcompositions can be made to the various embodiments of the inventiondescribed without departing from the spirit of the invention.

In one aspect the G-CSF compositions can be administered parenterally,for example, subcutaneously or intravenously. The parenteraladministration can be in a single bolus or as a continuous infusion. Inone aspect, the parenteral administration can be a single intravenousinfusion given over 15-30 minutes. In another aspect the parenteraladministration can be a continuous infusion of G-CSF diluted in 5%dextrose.

The methods provide for administration of G-CSF for a therapeutically orprophylactically effective time. In certain embodiments, the G-CSF isadministered prior to the onset or observation of the disorder orsymptoms accompanying the disorder. In further embodiments, the G-CSF isadministered during the disorder or during the time period that symptomsaccompanying the disorder are observed. In other embodiments, the G-CSFis administered for a time after the disorder had cleared. For example,the G-CSF can be administered about one day, about two days, about threedays, about four days, about one week, about two weeks and up to abouteight weeks, following resolution of threatened abortion or afterconfirmation of pregnancy during assisted reproduction.

Diagnostic Assays

While generally a medical history will serve to ascertain candidatesubjects in need of treatments as described above, diagnostic assays canbe used to ascertain subjects presented with reproductive inefficienciesthat are correlated with particular immunologic parameters. As notedherein, patients with repeated spontaneous abortion, miscarriage, orimplantation failure and the like present with particular profiles oftheir immune system status. Thus, subjects with high Th1 cell number orcell activity and/or reduced Th2 cell number or cell activity, or anaberrant ratio of the two may be candidates for obtaining theabove-described treatment.

Hence, a diagnostic assay of interest is one that determines whether Th1cell number or cell activity is enhanced. Another assay of interest isone that determines whether Th2 cell number or activity is decreased.Yet another assay of interest is one that determines a higher ratio ofTh1 cell number to Th2 cell number, or Th1 cell activity to Th2 cellactivity.

A number of known assays, for example, immunoassays or bioassays, can beused to make such determinations. For example, γ interferon, tumornecrosis factor alpha, tumor necrosis factor β, IL-2, IL-12, and IL-18are markers of Th1 cells. Thus, assays for one or more of suchcell-specific markers can provide the basis to conclude a higher thannormal Th1 status. As to Th2, IL-4, IL-5, IL-6, IL-10 and IL-13 areknown markers of that cell type. Thus, assays for one or more of suchcell-specific markers can provide the basis to conclude a higher thannormal Th2 status.

Diagnostic assays can be also used to ascertain subjects presenting withreproductive inefficiencies that are correlated with particularpathopysiologic parameters. Pathophysiologic markers could includemarkers of cell stress such as heat shock proteins, markers of oxidativestress such as nitric oxide and free radicals, markers of cell injurysuch as hepatic transaminases and creatine kinase, and markers of celldeath including caspase 1 & 3.

In other embodiments, a diagnostic kit can be used to ascertain subjectspresenting with reproductive inefficiencies that are correlated withserum or ovarian follicular fluid G-CSF concentrations. Such a kit wouldserve as a theranostic complimenting G-CSF as a therapeutic.

Kits

In another aspect, the present invention provides kits for carrying outthe methods of the invention. In certain embodiments, the presentinvention provides kits for preventing or reducing the likelihood ofspontaneous abortion or implantation failure during and followingartificial insemination The kits comprise one or more effective doses ofG-CSF along with a label or labeling with instructions on using theG-CSF to prevent or reduce the likelihood of spontaneous abortion orimplantation failure during and following artificial inseminationaccording to the methods of the invention. In certain embodiments, thekits can comprise components useful for carrying out the methods such asdevices for delivering the G-CSF. In certain embodiments, the kit cancomprise components useful for the safe disposal of devices fordelivering the G-CSF, e.g., a sharps container for used syringes.

In other embodiments, the present invention provides kits for preventingor reducing the likelihood of spontaneous abortion and implantationfailure in recipients of IVF, FET, ICSI, GIFT and ZIFT. The kitscomprise one or more effective doses of G-CSF along with a label orlabeling with instructions on using the G-CSF to prevent or reduce thelikelihood of spontaneous abortion, miscarriage or implantation failureduring and following IVF, FET, ICSI, GIFT and ZIFT. In certainembodiments, the kits can comprise components useful for carrying outthe methods such as devices for delivering the G-CSF. In certainembodiments, the kit can comprise components useful for the safedisposal of devices for delivering the G-CSF, e.g., a sharps containerfor used syringes.

In one embodiment, the G-CSF in the kit is formulated for subcutaneousadministration. In another embodiment, the G-CSF in the kit isformulated for intramuscular administration. In another embodiment, theG-CSF in the kit is formulated for intravascular administration.

The kit may further contain other active compounds, such as CSFs (e.g.,G-CSF, GMCSF, and macrophage CSF), erythropoietin, stem cell factors,anti-inflammatory agents, interleukins, etc.

In one embodiment, the G-CSF in the kit is contained in an implantabledevice for slow release after implantation of the device. In anotherembodiment, the G-CSF in the kit is contained in a transdermal patch forslow release after application of the transdermal device.

In another embodiment, the present invention provides a transdermalpatch comprising G-CSF as an active ingredient. In another embodiment,the present invention provides an implantable device comprising G-CSF asan active ingredient. In yet another embodiment, the present inventionprovides an implantable device G-CSF embedded in an inert matrix.

In another embodiment, the present invention provides a vaginal ringcomprising G-CSF as an active ingredient and in some cases with anothercomplimentary agent as an additional active ingredient.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

EXAMPLE 1 G-CSF Prevents Embryotoxic Effects of Cells From Women WithRecurrent Spontaneous Abortion In Vitro

G-CSF is effective in preventing the death of mouse embryos in an invitro clinical assay for spontaneous abortion. Mouse bioassays havewidely been used to detect embryotoxic effects of sera from subjectshaving reproductive difficulty. (See, Cameo, et al., 1999, Human Reprod.14 (4):959-63, Oksenberg and Brautbar 1986, Am. J. Reprod Immunol.Microbiol 11 (4):118-24, Roussev et al., 1995, Am. Reprod. Immunol. 33(2):171-175 and Thomason et al., 1995, Am. J. Reprod. Immunol. 34(6):338-41.).

In the in vitro clinical assay, mononuclear leukocytes are isolated fromwomen suffering from recurrent spontaneous abortion. The leukocytes arecultured, and the culture medium is removed from the leukocytes. Thisculture medium is then contacted with murine embryos. Toxic factors inthe culture medium typically kill the murine embryos in this assay.

The mononuclear leukocytes are incubated with G-CSF prior to removal ofthe culture medium. The culture medium is then removed from theleukocytes and contacted with murine embryos. Survival of the murineembryos indicates the reduction of embryotoxic factors in the culturemedium and thereby the effectiveness of G-CSF administration forprevention of spontaneous abortion in this in vitro model.

EXAMPLE 2 G-CSF Prevents Spontaneous Abortion In A Mouse Model In Vitro

G-CSF effectively inhibits a well-known in vivo model for spontaneousabortion.

The murine mating pair CBA×DBA/2 (see e.g., Yabuki et al., 2003, Exp.Anim. 52 (2) 159-63) results in a spontaneous abortion rate ofapproximately 40%. In this example, female CBA mice are treatedaccording to the methods of the invention. They are treated with G-CSFprior to mating, at the time of mating and immediately after mating. Areduction of the rate of spontaneous abortion in mice treated with G-CSFrelative to control mice indicates that G-CSF effectively preventsspontaneous abortion in this in vivo model.

EXAMPLE 3 G-CSF Prevents Habitual Abortion In Vivo

Thirty-one women with habitual abortion, having more than threeabortions, were recruited in the study (Scarpellini & Sbracia, 2004, Am.J. Repro. Imm. 51 (6) 433-4). The cytogenetic studies,hysterosalpingography, ultrasound, endometrial biopsy, hormonal assays(estradiol, progesterone, prolactin, thyroid hormones, etc.) diabetesworkup and autoantibody tests (ACA, AND, AMA, SMA and anti-lupus Ab)were unremarkable. All of the women failed a previous treatment withIgs, or corticosteroid and aspirin in the former pregnancy. Sixteenwomen were randomly chosen and treated with filgrastim at 100 mg/day SC,which was started the sixth day after ovulation and continued throughthe 35th day after ovulation. The other fifteen women received a placeboand progesterone.

In the group receiving filgrastim, 14 of 16 became pregnant andmaintained pregnancy during the recording period. The karyotype of thefetuses was normal. In the control group, only four pregnanciesoccurred. hCG levels in the treated women were increased by a third overthe levels observed in the control women.

Over the course of the last 4 years, three patients undergoing assistedreproduction procedures have been treated with recombinant hG-CSF(rhG-CSF). Case studies of these three patients are provided below.

(1) J. C.

J. C. is a 36-year-old married white female with an obstetrical historyof three uncomplicated vaginal deliveries at full term (all malechildren) followed by six consecutive first trimester miscarriages (eachat 10-12 weeks). Conception was natural in each of the successfulpregnancies and in each miscarriage. Each miscarried fetus waskaryotyped, and all were normal. The couple then experienced three yearsof secondary infertility. At that point, she sought a consultation witha reproductive endocrinologist (RE).

The RE performed a detailed workup to attempt to identify the cause ofthe couple's reproductive failures. No anatomic or endocrinologicetiology was identified. Both J. C. and her husband were found to bekaryotypically normal. A standard andrology workup for the husband wasnegative.

J. C.'s past medical history was significant in that J. C. had a remotepast history of seasonal allergies and ten years of allergydesensitization shots. Based on this medical history, a series ofimmunologic tests including measurement of Th1 and Th2 cytokineproduction in vitro were ordered. As noted previously in thisapplication, allergy is a classic Th2 immunopathologic response.Although few allergists realize it, allergy desensitization works bypresenting the allergen in a manner that favors Th1 cytokine productioninstead of Th2 cytokine production. In many individuals, this shift fromTh2 to Th1 dominance becomes more generalized and antigen non-specific.The series of tests ordered for J. C. specifically measured Th1/Th2cytokines produced by the patient's peripheral blood mononuclear cells(PBMC) in response to the non-specific mitogen phytohemagglutinin (PHA).J. C.'s PBMC produced greater than 10,000 units per ml of the prototypicTh1 cytokine gamma interferon in response to PHA. Levels of theprototypic Th2 cytokine IL-4 and the counter regulatory Th2 cytokineIL-10 were undetectable.

The RE performed intrauterine insemination (IUI) using J. C.'s husband'ssperm. The first attempt at IUI resulted in a positive HCG at 7 days.The rhG-CSF administration was initiated the following day. The regimenconsisted of 100 mcg/day of rhG-CSF (Neupogen) injected subcutaneouslyfor a total of 30 days, a cumulative dose of 3000 mcg. The rhG-CSFregimen was carried out for the full 30 days and then discontinued. Thepatient experienced no rhG-CSF-related side effects at any point duringthe regimen.

At day 14 of the rhG-CSF regimen, another blood sample was obtained fromJ. C. for repeat measurement of Th1 and Th2 cytokines by her PBMC inresponse to PHA. The repeat results showed undetectable levels of theprototypic Th1 cytokine gamma interferon and elevated levels (2,000units per ml) of the counter regulatory Th₂ cytokine IL-10. Theseresults clearly indicated that rhG-CSF produced a shift from Th1 to Th2cytokine production by her PBMC in response to PHA. Interestingly, J.C.'s allergies had also returned. This is consistent with the shift fromTh1 to Th2 cytokine dominance.

At 8 weeks, an ultrasound confirmed an ongoing healthy pregnancy with awell-formed gestational sac and a fetus with a strong heartbeat. Thepregnancy continued to progress uneventfully and at 11 weeks J. C. wastransferred from the care of her RE to the care of a generalobstetrician. The pregnancy progressed without complication, and ahealthy 8 lb., 19-inch female was delivered by planned cesarean sectionat 38 weeks. Mother and child are both doing well.

(2) N. C.

N. C. is a healthy 35-year-old married white female with an obstetricalhistory of primary infertility including three failed IUIs and onefailed IVF.

N. C.'s first IUI resulted in monozygotic twins, one of which revealedno fetal heartbeat at 6 weeks and the other which had a confirmed weakfetal heartbeat at 6 weeks but no heartbeat by the 7^(th) week. Thesecond IUI resulted in a singleton pregnancy and fetal demise at 8weeks. A heartbeat was seen at the 7^(th) week but was negative by the8^(th) week. Karyotyping was performed and revealed an abnormalkaryotype (69 XXY). N. C.'s third IUI resulted in a probable ectopicpregnancy treated with methotrexate. N. C.'s last pregnancy attempt wasa cycle of IVF. This resulted in a confirmed and apparently healthypregnancy at 6 weeks with a gestational sac measuring 36×37 millimetersand fetal heart rate of 113. However, one week later no fetal heartbeatwas observed. The products of conception were expelled in large clots,and karyotyping was performed. Karyotyping was revealed to be normal (46XY). N. C.'s RE performed an exhaustive workup to determine the cause ofher reproductive failures. However, the workup failed to reveal anyidentifiable cause.

N. C.'s past medical history was non-contributory. She appeared to be ahealthy female with unexplained primary infertility and repeatedpregnancy loss. A review of her medical records revealed past laboratorytesting showed a normal balance of Th1 and Th2 cytokines.

Because one of N. C.'s early losses involved a karyotypically abnormalembryo (69 XXY), N. C. had arranged for preimplantation geneticdiagnosis for her last (failed) IVF cycle. N. C. had two cryopreservedembryos left from that cycle, and those embryos were used for the IVFcycle with rhG-CSF. N. C. received 100 mcg per day for the seven daysprior to transfer and for 30 additional days after transfer, at acumulative dose of 3700 mcg. N. C. experienced no rhG-CSF related sideeffects. At 6 weeks an ultrasound evaluation of N. C. revealed a healthypregnancy with a well-formed gestational sac (40×40 mm) and a strongheart beat (145 beats per minute). At the 10th week, N. C. wastransferred from her RE's care to the high-risk obstetrical unit in ahospital where she delivered a healthy baby boy. Both mother and childare doing well.

Approximately one year later, N. C. opted to undergo another IVF cycleat a different clinic without the benefit of rhG-CSF therapy. This cyclefailed and was classified as a biochemical pregnancy (positive beta HCG,no evidence of gestational sac or embryo).

A few months later, N. C. contacted the inventor to request that heprovide consultation regarding the use of rhG-CSF in her next IVF cycle.The inventor agreed and a clinical plan identical to her previous IVFcycle using rhG-CSF was pursued. N. C. began rhG-CSF (100 mcg per day)five days prior to embryo transfer (i.e., on the day of oocyteretrieval) in a fresh IVF cycle. The pregnancy is ongoing and her RE hastransferred her to the care of a general obstetrician. At her lastexamination (at 20 weeks), all measurements were normal for gestationalage and fetal heartbeat was strong.

(3) J. J.

J. J. is a 33-year-old married white female with a history of primarysubfertility and seven failed pregnancies. Over a period of three years,J. J. suffered three first-trimester miscarriages and three chemicalpregnancies. Four of the pregnancies involved the use of fertility drugsand natural conception. Two of the pregnancies occurred through IUI. Thelast pregnancy was a failed cycle of IVF.

J. J.'s RE performed a standard workup to attempt to determine cause forJ. J.'s failures. The workup failed to identify a cause. Both members ofthe couple were found to be karyotypically normal. J. J. and her REdecided that she should consult with a Reproductive Immunologist. Priorto J. J.'s IVF cycle, this physician performed a battery of laboratorytests and a medical evaluation and concluded that J. J. should undergo acourse of Intravenous Immunoglobulin (IVIG) to correct immune problemsidentified through testing. Repeat laboratory tests demonstrated thatIVIG failed to correct the purported immunologic problem. J. J.'s IVFcycle resulted in an ectopic pregnancy, and J. J. required emergencysurgery for a unilateral salpingectomy.

J. J. and her RE sought a consultation with the inventor and decided toundergo another cycle of IVF with rhG-CSF treatment.

J. J. underwent another cycle of IVF with frozen embryos from herprevious cycle. Although J. J. was scheduled to begin rhG-CSF at 100 mcgper day five days prior to embryo transfer, J. J. was not able to beginrhG-CSF until three days before embryo transfer. The rhG-CSF wascontinued at 100 mcg per day for 30 days after embryo transfer. Thecumulative dose of rhG-CSF was 3300 mcg. J. J. completed her course ofrhG-CSF and experienced no rhG-CSF related side effects.

Two embryos were transferred. The cycle resulted in a positive beta HCG(139 at 7 days post transfer; 316 at 10 days post transfer). Six weekspost transfer, an ultrasound identified a well-formed gestational sacand a heart beat of 115.

J. J. underwent another ultrasonic evaluation at 10 weeks gestation, anda strong heartbeat was identified and all measurements were exactlyappropriate for dates. J. J. was transferred to the care of a generalobstetrician and delivered a healthy baby girl. Both the mother and thechild are healthy and doing well.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1. A method for preventing or reducing the likelihood of implantationfailure or miscarriage in a recipient of artificial insemination,comprising administering to the recipient of artificial insemination aneffective amount of a composition comprising G-CSF.
 2. The method ofclaim 1, wherein said artificial insemination is intrauterineinsemination, intravaginal insemination, intracervical insemination, orintratubal insemination.
 3. The method of claim 1, wherein saidcomposition is administered parenterally.
 4. The method of claim 1,wherein said composition is administered enterally.
 5. The method ofclaim 1, wherein said composition is administered topically.
 6. Themethod of claim 1, wherein said composition is administered byinhalation.
 7. The method of claim 1, wherein said composition isadministered prior to said artificial insemination.
 8. The method ofclaim 1, wherein said artificial insemination comprises a controlledovarian hyperstimulation procedure prior to insemination, and whereinsaid composition is administered before, during, or after the time ofcontrolled ovarian hyperstimulation.
 9. The method of claim 1, whereinsaid composition is administered daily for one to thirty-fiveconsecutive days.
 10. The method of claim 1, wherein said composition isadministered daily until the end of first trimester.
 11. The method ofclaim 1, wherein said composition is administered daily until saidrecipient presents a normal Th1 response or a normal Th2 response orboth.
 12. The method of claim 1, wherein said G-CSF is administered at adose of between 0.1 mcg/kg/day to 600 mcg/kg/day.
 13. The method ofclaim 12, wherein said G-CSF is administered at a dose of between 1mcg/kg/day to 100 mcg/kg/day.
 14. The method of claim 13, wherein saidG-CSF is administered at a dose of between 1 mcg/kg/day to 50mcg/kg/day.
 15. The method of claim 14, wherein said G-CSF isadministered at a dose of between 1 mcg/kg/day to 10 mcg/kg/day.
 16. Themethod of claim 15, wherein said G-CSF is administered at a dose ofbetween 1 mcg/kg/day to 2 mcg/kg/day.
 17. The method of claim 16,wherein said G-CSF is administered at a dose of about 1.5 mcg/kg/day to1.7 mcg/kg/day.
 18. The method of claim 1, wherein said G-CSF isadministered in the form of a nucleotide sequence encoding G-CSF. 19.The method of claim 1, wherein said composition further comprises anadditive.
 20. The method of claim 19, wherein said additive is selectedfrom the group consisting of cytokines that suppress Th1 immuneresponse, cytokines that enhance Th2 immune response, cytokines thatsupport successful pregnancy through non-immunologic mechanisms,anti-inflammatory agents, and inhibitors of pro-inflammatory cytokines.21. The method of claim 19, wherein said additive is selected from thegroup consisting of interferon alpha, interferon beta, macrophage colonystimulating factor (M-CSF), granulocyte macrophage colony stimulatingfactor (GM-CSF), leukemia inhibitory factor (LIF), transforming growthfactor beta (TGF-beta), interleukin-1 (IL-1), IL-3, IL-4, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-13, IL-14, IL-15, IL-19, IL-20, IL-21,IL-22, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32,IL-33, and IL-35.
 22. The method of claim 19, wherein said additivecomprises an immunosuppressive agent.
 23. The method of claim 22,wherein said immunosuppressive agent comprises vitamin D3 (1,25dihydroxycholecalciferol), analogs thereof, or corticosteroids.
 24. Themethod of claim 23, wherein said corticosteroid is prednisone ormethylprednisolone.
 25. The method of claim 1, wherein said compositionfurther comprises a pharmaceutically acceptable carrier.
 26. A kit forpreventing or reducing the likelihood of implantation failure andmiscarriage in a recipient of artificial insemination, comprising aneffective amount of G-CSF; and a label with instructions for using theG-CSF to prevent or reduce the likelihood of implantation failure andmiscarriage.
 27. The kit of claim 26, wherein said G-CSF is formulatedfor subcutaneous administration.
 28. The kit of claim 26, wherein saidG-CSF is formulated for intramuscular administration.
 29. The kit ofclaim 26, wherein said G-CSF is formulated for intravascularadministration.
 30. The kit of claim 26, further comprising at least oneother active compound.
 31. The kit of claim 30, wherein said otheractive compound is another CSF, erythropoietin or stem cell factor. 32.The kit of claim 31, wherein said CSF is G-CSF, GMCSF or macrophage CSF.33. The kit of claim 30, wherein said other active compound is ananti-inflammatory agent.
 34. The kit of claim 30, wherein said one otheractive compound is an interleukin.
 35. The kit of claim 26, wherein saidG-CSF is contained in an implantable device for slow release afterimplantation of the device.
 36. The kit of claim 26, wherein said G-CSFis contained in a transdermal patch for slow release after applicationof the transdermal device.
 37. The kit of claim 26, wherein said G-CSFis contained in a vaginal ring.
 38. An implantable device, comprisingG-CSF embedded in an inert matrix.